This grant continues to be aimed at understanding how actin filaments interact with the plasma membrane, concentrating on the attachment of actin filament bundles at focal contacts and at sites of adhesion to the extracellular matrix. During the last few years we have identified several new components in these regions and have established some of the interactions that occur between them. We have demonstrated an interaction between vinculin and talin, and between talin and the cytoplasmic domain of fibroblast integrin. How actin filaments associate with these other focal contact components remains an unanswered question. The interaction between talin and platelet plasma membrane vesicles will be studied, focussing on the possible mediation of this association by the platelet form of integrin, glycoproteins IIb/lIla. Although we have been unable to establish a direct interaction between talin and actin, indirect evidence suggests that talin may regulate actin polymerization in some circumstances. We will investigate whether talin can affect actin polymerization via the proteins gelsolin and profilin. Preliminary evidence indicates that vinculin may interact with several proteins, some of which have not been purified. We will attempt to purify these components and to characterize their interactions with vinculin and other focal contact constituents. We have found that integrin purified from smooth muscle has actin bound to it. This association will be investigated to determine whether it is physiologically relevant and whether integrin will nucleate actin polymerization. The role of integrin in focal contacts will be examined in live cells by microinjection of peptides corresponding to integrin's cytoplasmic domains. Do these peptides disrupt stress fiber attachment, perturb focal contact organization or cause cell rounding? Focal contact membranes will be prepared from fibroblasts or platelets spread on coverslips. The extraction of specific components and their reassociation with the stripped membranes will be studied, as will the conditions required for these focal contact membranes to nucleate actin polymerization. The development of a model system such as this should help to define the functions of different components in these sites of actin-membrane attachment. Finally, we will search for new focal contact components by immunizing mice with crude focal contact preparations or extracts of smooth muscle, developing monoclonal antibodies and screening these for focal contact staining. New components identified by these antibodies will be purified and characterized.